Aberrant uracils in human genomic DNA lead to G:C to A:T transversion mutation, the most frequent mutation found in many cancers. Human uracil DNA glycosylase (hUNG) searches and removes uracil bases from deoxyribose phosphate backbone using a remarkable search mechanism that allows the enzyme to rapidly locate rare lesion sites in a background of roughly 6 billion normal base pairs in the human genome. This general search mechanism has been referred to as facilitated diffusion, where proteins utilize the DNA chain as a track to accelerate location of their specific sites. Facilitated diffusion uses two microscopic transfer pathways: one-dimensional sliding and three-dimensional hopping. Although numerous studies have established the existence of such a general mechanism, the details of facilitated diffusion remain mysterious. The focus of this proposal is to elucidate th molecular interactions that are important for DNA chain tracking by hUNG. Our recently developed molecular clock (MC) approach utilizes a small molecule trap to capture transient hopping enzymes while having minimal effect on the sliding ones, thus allowing the dissection of the two tracking pathways individually. Key parameters that can be probed using this approach include the calculation of mean sliding distance, the average distance an enzyme hops away from DNA, and the 1D diffusion constant for DNA sliding. In the first aim, we will use MC approach to test several key aspects of DNA tracking. First, to test the role of DNA phosphate electrostatics, we will insert neutral methylphosphonate linkages in the DNA backbone between two uracil target sites separated by a set number of base pairs, and then measure the effect of charge ablation on the probability that hUNG will slide between the sites. Second, we will test a novel hypothesis that directionally-biased transfer can occur (even in the absence of an energy- providing cofactor) if thermodynamically stable binding sites are inserted between the two uracil target sites. This aim will involve the insertion of spaced high-affinity tetrahydrofuran abasic sie analogues between the uracil sites. The prediction is that a biased walk will occur via high-affinity island transfer. A biased-walk using basic sites is highly relevant to the in vivo situaion where hUNG must locate and excise clustered uracil sites such as in the process of Ig somatic hypermutation. The second aim is to engineer hUNG-peptide tail variants to have enhanced tracking abilities. Fusion proteins between hUNG and short, positively charged peptides will be constructed using expressed protein ligation technology (EPL) and/or chemical ligation. These peptides will be derived from several DNA binding proteins (p53, HOXD9, H3), and are chosen because they represent various sizes and overall charge, and are known to affect DNA binding and association kinetics. We will measure the fundamental tracking parameters with these variants, which will directly test how the residence time on nonspecific DNA, and the 1D diffusion constant for sliding affect the efficiency of damage repair both in vitro and in vivo.